Exploring endophyte-mediated resistance response against winter cutworm, Noctua pronuba in cool-season turfgrass systems

This study evaluated host plant resistance in terms of winter cutworm feeding response in no-choice assays using commercial perennial ryegrass and tall fescue cultivars with varying endophyte infection levels. 

1. Assess winter cutworm survivorship and development time on previously identified cool-season turfgrass cultivars in Oregon State University’s NTEP and A-List trials containing a viable endophyte.
2. Validate the endophyte-mediated resistance response of promising cultivars to winter cutworms by conducting a series of lab experiments whereby endophytic fungi are removed in known E+ (endophyte-positive) turfgrass cultivars and inoculated in E- (endophyte-negative) turfgrass cultivars.

   Cultivars of tall fescue with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified as outlined in Obj 1. However, these cultivars were unavailable commercially, so the proposed objectives (listed above) were modified to conduct two no-choice trials in the greenhouse conditions using four cultivars of two grass species (tall fescue, perennial ryegrass), two levels of endophyte infection (high, low), and two fungicide treatments (no fungicide, fungicide), along with simultaneous winter cutworm (N. pronuba) feeding (winter cutworm, control).

Procedures:

Two no-choice trials were conducted between August and November 2023 at the West Greenhouses, Oregon State University, Corvallis, OR.

Insect collection and rearing method

Winter cutworm, N. pronuba, eggs were collected from commercial grass seed fields in Oregon, USA (45°11'14.4"N 123°14'59.7"W and 44°33'12.3"N 123°18'02.8"W) from August to September 2023. Eggs were checked for parasitism before the introduction into a laboratory for rearing. Healthy eggs and collections without any prior exposure to insecticides were used in the laboratory colonies. The insect colonies were maintained on a general-purpose lepidopteran diet (Frontier Scientific Inc., Newark, DE) using a method slightly modified (Hervet et al. 2016) under a laboratory condition (20 ± 2 °C; 9:15 (L:D) h photoperiod; 70% humidity). One liter of diet was prepared as per the protocol modified from the recipe provided by Frontier Scientific. 875 ml of DI water was boiled in a beaker on a hotplate with a stirring bar, after which 105 g of dry mix were slowly added to the beaker. A rubber spatula was used to vigorously mix the slurry to prevent burning on the bottom of the beaker. Once the diet was fully mixed, 19 g of agar was added to the mixture, followed by vigorous mixing for five minutes. The diet was then transferred into 1 oz-cups, filling them up to ¼ of their volume. 

Plants and seed sources

Cultivars of tall fescue and perennial ryegrass with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified. Two cultivars of each grass species-endophyte combination (e.g. tall fescue-high endophyte, tall fescue-low endophyte, perennial ryegrass-high endophyte, perennial ryegrass-low endophyte) were used in the experiment.

Seeds of the eight cultivars of both grass species were obtained from five different seed storage and distribution facilities in Oregon during Spring 2023, as detailed in Table 1 . The seeds were stored at 4°C when not in use. These seeds were sown in a 40-cell tray insert (150 cm³ per cell) containing RESiLIENCE® potting mix (comprising 35-45% Canadian sphagnum peat moss, processed softwood bark, perlite, coir, and dolomite) (Sun Gro Horticulture, Agawam, MA). Five seeds per cultivar were sown in each cell at a depth of 1 cm. One week after germination, seedlings were manually thinned to one per cell. The grasses were maintained under a 16:8h L:D photoperiod with supplemental lighting from 1000 W sodium vapor bulbs and watered as needed. Day and night temperatures were set at 25 and 20°C, respectively. Another set of the same seeds was treated with a systemic fungicide (Banner Maxx II) containing propiconazole as the active ingredient (Syngenta, Wilmington, DE) to create fungicide-treated counterparts. The seeds were soaked in the fungicide solution (0.5 g a.i .) for 25 min and air-dried on paper towels overnight. The appropriate fungicide rate for controlling endophytes in seedlings was previously determined. Fungicide-treated seeds were planted and maintained in the greenhouse as described above.   

Insect survivorship and development

Eight to nine weeks after the seeds germinated, five plant plugs of each cultivar-fungicide treatment combination were transferred into a 5-gallon plastic storage tote filled with potting mix. The container bases and lids were drilled with six (size) and two holes (size), respectively, to enhance ventilation. Meshes were glued over the holes on the lid and placed between the base and potting mix to prevent larval escape. The potting mix consisted of a 1:1 ratio of G&B Simples Seed Starter Mix (comprising peat moss, perlite, pumice, washed sand) (Kellogg Garden Products, Carson, CA) and autoclaved kiln-dried sand (Marion Ag Services, Inc., St. Paul, OR).

Five second to third instar winter cutworms (with a total weight ranging from 0.14 – 0.55g for trial 1 and 0.33 – 1.13g for trial 2) were added to each plastic container containing five transferred test plants. Every three days after the insect release, the numbers of observed live and dead larvae were recorded, and the aboveground grass area was photographed. The winter cutworm larvae were allowed to feed on the grasses for 14 days before removing them from the arena. Missing larvae were considered dead, and all live larvae were weighed. Each plant was cut at ground level and weighted before three representative tillers were taken and stored in an Agdia’s mesh sample bag at -20°C for endophyte detection using molecular methods (Schardl et al., 2012; Vivuk et al., 2019) (Table 1). This experiment was repeated twice.

Molecular endophyte determination methods

Total nucleic acids were extracted from frozen plant material using the protocol modified from Dellaporta et al. (1983). The modification involved the use of drill press attached with an Agdia tissue homogenizer (Agdia, Inc., Elkhart, IN) to grind samples in the Agdia’s mesh sample bags. The endophyte infection status, infection rate, and chemotype were determined using a high throughput multiplex PCR method described by Takach et al. (2012). The translation elongation factor 1-alpha (tefA) and the following alkaloid biosynthesis-related genes: peramine synthetase (perA), lolC, dmaW, and idtG were amplified from total DNA. All genes have been widely used for endophyte detection and chemotypic analysis of endophytes worldwide (Schardl et al., 2013). The primers used for the multiplex PCR reaction are listed in Table 2. The PCR reactions with a total volume of 25 µl each contained 5× Green GoTaq reaction buffer, 10 μM of each deoxynucleoside triphosphate (dNTP), 1.0-U GoTaq DNA polymerase (Promega Corp., Madison, WI), and each target-specific primer (10 µM). Amplification conditions were two min of initial denaturation at 94°C, 30 cycles of 15s at 94°C, 30s at 56°C, and one min at 72°C, followed by seven min at 72°C (Takach et al., 2012). PCR products were visualized via gel electrophoresis with a 1.5% agarose gel in 1X Tris-borate-EDTA (TBE) buffer following ethidium bromide staining and UV transillumination. The expected fragment sizes for each amplicon was presented in Table 2. Each chemotype was characterized using banding patterns.

Table 1 seed source and cultivar profiles

Table 1. Primers used in the multiplex PCR.

Representative PCR products were purified with the PCR purification kits and sent for sequencing to generate sequence data of Epichloë strains (wild type or novel) among promising cultivars identified during the insect-feeding bioassays.